Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
2.1 Fungi and Disease
Fungi are eukaryotic microorganisms comprising a phylogenetic kingdom. The Kingdom Fungi is estimated to contain over 100,000 species and includes species of “yeast”, which is the common term for several families of unicellular fungi.
Although fungal infections were once unrecognized as a significant cause of disease, the extensive spread of fungal infections is a major concern in hospitals, health departments and research laboratories. According to a 1988 study, nearly 40% of all deaths from hospital-acquired infections were caused by fungi, not bacteria or viruses (Sternberg, S., 1994, Science 266:1632–34).
Immunocompromised patients are particularly at risk for fungal infections. Patients with impaired immune systems due to AIDS, cancer chemotherapy, or those treated with immunosuppressive drugs used to prevent rejection in organ transplant are common hosts for fungal infections. Organisms including but not limited to Cryptococcus spp., Candida spp., Hostoplasma spp., Coccidioides spp., and as many as 150 species of fungi have been linked to human or animal diseases (Sternberg, S., 1994, Science 266:1632–34). Under immunocompromised conditions, fungi that are normally harmless to the host when maintained in the gastrointestinal system, can be transferred to the bloodstream, eyes, brain, heart, kidneys, and other tissues leading to symptoms ranging in severity from white patches on the tongue, to fever, rupturing of the retina, blindness, pneumonia, heart failure, shock, or sudden catastrophic clotting of the blood (Sternberg, S., 1994, Science 266:1632–34). In susceptible burn victims, even S. cerevisiae (baker's yeast), common in the human mouth and normally non-virulent, can lead to severe infection (Sternberg, S., 1994, Science 266:1632–34). Hospital transmission may also occur via catheters or other invasive equipment (Sternberg, S., 1994, Science 266:1632–34).
Fungal infections are not limited to individuals with compromised immune systems. Geological and meterological events have been reported to trigger fungal outbreaks. Following a 1994 earthquake in California, tremors were estimated to have released infectious fungal pores from the soil triggering a 3-year statewide epidemic that led to more than 4,500 cases per year (Sternberg, S., 1994, Science 266:1632–34).
Moreover, fungal infections are not limited to humans. Animals and plants are both struck by fungal infections. The worldwide contamination of foods and feeds with mycotoxins, the secondary metabolites of fungi, is a significant problem that has adverse effects on humans, animals and crops and results in substantial illness and economic loss. (Hussein, H. S. and Brasel, J. M., 2001, Toxicology:167(2):101–34). The economic impact of mycotoxins include loss of human and animal life, increased health care and veterinary care costs, reduced livestock production, disposal of contaminated foods and feeds, and investment in research and applications to reduce severity of the mycotoxin problem. (Hussein, H. S. and Brasel, J. M., 2001, Toxicology:167(2):101–34). Clearly, efforts to control the spread of fungi will concomitantly control the often costly byproducts of fungi, mycotoxins.
The widespread dissemination of fungal infection coupled with the recognition of fungi as a significant disease causing factor creates an increasing need for antifungal agents. Existing antifungal therapies harbor many disadvantages as discussed below in Section 2.2, and novel antifungal agents and therapies are needed.
2.2 Antifungal Agents and the Need for Improvement
An effective antifungal agent is toxic to the pathogenic fungi, but not to the host. One way to achieve this goal is to target a structure or pathway that is unique to the pathogen. For example, successful antibacterial therapies often take advantage of the differences between the prokaryotic bacteria and the eukaryotic host. However, since fungal pathogens, like human cells, are eukaryotic, it has been more difficult to identify therapeutic agents that uniquely affect the pathogen. A lack of sufficient pathogen specificity can result in host toxicity. Treatment of fungal diseases is often limited because antifungal agents are often toxic to the mammalian or plant host, frequently resulting in severe side effects. For example, the commonly prescribed drug, Amphotericin B, a mainstay of antifungal therapy, includes such side effects as fever, chills, low blood pressure, headache, nausea, vomiting, inflammation of blood vessels and kidney damage (Stemberg, S., 1994, Science 266:1632–34). Further, many of the existing therapies act to inhibit or slow fungal growth, but do not kill the infecting fungi.
Currently, there are five main classes of antifungal compounds: azoles; polyenes; allylamines; flucytosine; and candins. Each class is characterized by its mode and/or site of action. Azoles inhibit the synthesis of ergosterol, the main fungal sterol. Polyenes bind to fungal membrane sterol, resulting in the formation of aqueous pores through which essential cytoplasmic materials leak out. Allylamines block ergosterol biosynthesis, leading to accumulation of squalene, which is toxic to cells. Flucytosine inhibits macromolecular synthesis. Finally, candins inhibit the synthesis of 1,3-β-glucan, the major structural polymer of the fungal cell wall, thereby inhibiting fungal growth. (Balkis, M. M., et al., 2002, Drugs 62(7):1025–40).
Additionally, the increased use of antifungal agents in recent years has resulted in the development of fungal resistance to these drugs. The prospect of acquired resistance in fungal pathogens to known antifungal agents is likely to continue to fuel the search for novel and more effective antifungal agents.
2.3 The Cell Wall and the Glucan Synthase Pathway
The fungal cell wall is a complex, dynamic network whose structure and function are both unique and essential to fungal cell life and development. The fungal cell wall thus serves as an ideal target for antifungal agents. In addition to helping a cell maintain its shape and protecting the cell against osmotic forces, the cell wall acts as a filter, controlling uptake and secretion of molecules into and out of the cell. (Wills, E. A., et al., 2000, Emerging Therapeutic Targets 4(3):1–32). Interference with fungal cell wall function, structure or synthesis will eventually lead to cell lysis and death. (Wills, E. A., et al., 2000, Emerging Therapeutic Targets 4(3):1–32).
The fungal cell wall comprises a meshlike structure of polysaccharides, including 1,3-β-glucan, 1,6-β-glucan, and chitin. (Douglas, et al., 1994, J. Bacteriology 176(18):5686–5696). Significantly, 1,3-β-glucan is the most prominent carbohydrate component of the fungal cell wall. (Wills, E. A., et al., 2000, Emerging Therapeutic Targets 4(3):1–32). Thus, the membrane-bound enzyme which catalyzes the synthesis of 1,3-β-glucan, the enzyme glucan synthase (EC 2.4.1.34 [UDP-glucose: 1,3-β-D-glucan 3-β-glucose transferase]), plays an indispensable role in cell wall biosynthesis. (Douglas, et al., 1994, J. Bacteriology 176(18):5686–5696). Specifically, glucan synthase transfers glucose from UDP-glucose to an acid-insoluble, alkali-soluble, exo-β-1,3-glucan-sensitive polysaccharide. This fundamental role, coupled with the fact that glucan synthase is not found in mammalian cells, makes the glucan synthase pathway an ideal target for antifungal agents. Several known antifungal agents, such as Enfumafungin, Ascosteroside, and dihydropapulacandin, act by inhibiting the glucan synthase pathway. (Gorman, J. A., et al., 1995, J. Antibiotics, 49(6):547–52).
Similarly, it is known that disrupting S. cerevisiae glucan synthase pathway genes FKS1 and/or FKS2 results in cell wall damage. (Terashima, H., et al., 2000, Mol. Gen. Genet. 264:64–74). FKS1 and FKS2 encode alternative catalytic subunits of the glucan synthases that are responsible for the synthesis of 1,3-β-glucan. (Terashima, H., et al., 2000, Mol. Gen. Genet. 264:64–74).
Furthermore, a glucan synthase complex or a homologous glucan synthase gene has been documented in the following pathogenic fungal species: Saccharomyces cerevisiae (Inoue, S. B., et al, 1995, Eur. J. Biochem. 231:845–854); Candida albicans (Mio, T., et al., 1997, J. Bacteriol., 179:4096–4105); Schizosaccharomyces pombe (Arellano, M, et al., 1996, Embo. J. 15:4584–4591); Aspergillus nidulans (Kelly, R., et al., 1996, J. Bacteriol. 178: 4381–4391); Neurospora crassa (Awald, P., et al., 1994, Biochim. Biophys. Acta 1201(2):312–320); and Cryptococcus neoformans (Thompson, J. R., et al., 1999, J. Bacteriol. 181(2):444–453).
At present, there is a need in the art for efficient and economical methods to evaluate potential antifungal molecules for their effect on the glucan synthase pathway. Current methods of screening for novel glucan synthase pathway inhibitors include in vitro screening assays for molecules that inhibit polymerization by glucan synthase.
Current methods however, harbor several disadvantages and shortcomings. The primary drawbacks of the in vitro assay are its difficulty to perform and the possibility that molecules which inhibit polymerization by glucan synthase in vitro, may not have that effect in vivo. The methods described in the instant invention can easily be assayed in a non-invasive fashion that is suitable to a broader spectrum of assay conditions and is suitable to high-throughput assays.
2.4 Microarray Technology
Within the past decade, several technologies have made it possible to monitor the expression level of a large number of transcripts at any one time (see, e.g., Schena et al., 1995, Quantitative monitoring of gene expression patterns with a complementary DNA micro-array, Science 270:467–470; Lockhart et al., 1996, Expression monitoring by hybridization to high-density oligonucleotide arrays, Nature Biotechnology 14:1675–1680; Blanchard et al., 1996, Sequence to array: Probing the genome's secrets, Nature Biotechnology 14, 1649; U.S. Pat. No. 5,569,588, issued Oct. 29, 1996 to Ashby et al entitled “Methods for Drug Screening”). In organisms for which the complete genome is known, it is possible to analyze the transcripts of all genes within the cell. With other organisms, such as humans, for which there is an increasing knowledge of the genome, it is possible to simultaneously monitor large numbers of the genes within the cell.
Such monitoring technologies have been applied to the identification of genes which are up-regulated or down-regulated in various diseased or physiological states, the analyses of members of signaling cellular states, and the identification of targets for various drugs. See, e.g., Friend and Hartwell, International Publication WO98/38329 (dated Sep. 3, 1998); Stoughton and Friend, U.S. Pat. No. 5,965,352 (issued on Oct. 12, 1999); Friend and Hartwell, U.S. Pat. No. 6,165,709 (issued on Dec. 26, 2000), U.S. Pat. No. 6,324,479 (issued on Nov. 27, 2001), all incorporated herein by reference for all purposes.
Levels of various constituents of a cell are known to change in response to drug treatments and other perturbations of the cell's biological state. Measurements of a plurality of such “cellular constituents” therefore contain a wealth of information about the effect of perturbations and their effect on the cell's biological state. Such measurements typically comprise measurements of gene expression levels of the type discussed above, but may also include levels of other cellular components such as, but by no means limited to, levels of protein abundances, or protein activity levels. The collection of such measurements is generally referred to as the “profile” of the cell's biological state.
The number of genes in a S. cerevisiae cell is typically on the order of more than 6,000 genes. The profile of a particular cell is therefore typically of high complexity. Any one perturbing agent may cause a small or a large number of cellular constituents to change their abundances or activity levels. Thus, identifying the particular cellular constituents which are associated with a certain biological pathway, such as the glucan synthase pathway, provides a difficult and challenging task.
In order to efficiently monitor and study a particular biological pathway, it is necessary to have a “read-out” or reporter of the pathway which allows measurement of an alteration of the pathway. Many biological pathways, however, do not have reliable reporters associated with them. Therefore, there is a need in the art to identify reporter genes, which are associated with a particular biological pathway. The present invention provides such reporter genes and methods of using such reporters to monitor the state of the glucan synthase pathway in S. cerevisiae and additionally, methods of using those reporter genes to screen chemical libraries and natural products for novel antifungal agents.